Project Summary/Abstract Bone loss diseases, including osteoporosis, are a significant and increasing threat for America's aging population. Degenerative osteopenia is a complex trait with environmental and genetic components, and may have arisen from a reduction in the strength of natural selection to maintain robust bone production in post- reproductive individuals. Natural variation for this complex trait exists in certain vertebrate lineages leading to the adaptive evolution of secondary osteopenia. We apply the innovative strategy of evolutionary mutant models for human disease to the skeletons of osteopenic Antarctic fish, whose ancestors possessed robust skeletons. As natural selection for dense bones diminished in certain lineages of Antarctic fish, the skeleton became osteopenic, allowing animals to inhabit the water column and exploit its abundant resources. Related lineages that retain dense skeletons continue to forage on the ocean floor. The goal of the proposed work is to characterize the genetic and phenotypic differences between species with osteopenic and normal skeletons, and thereby identify new candidate genes and mechanisms for human bone degeneration diseases. Our hypothesis is that mutations that either down-regulate the activity of genes that positively regulate osteogenesis or up-regulate the activity of genes that negatively affect osteogenesis account for evolved differences in related species with osteopenic versus robust skeletons. Aim 1 will identify the stages at which skeletal development diverges between the osteopenic species Chaenocephalus aceratus (blackfin icefish) and the related robustly ossified species Notothenia coriiceps (yellowbelly rockcod) using stains for cartilage, bone, and extracellular matrix molecules, and the expression of skeletal marker genes. Aim 2 will use high- throughput cDNA sequencing to compare gene expression profiles of skeletogenic tissues from densely and poorly ossified species as a means to identify regulatory differences between the two species. Aim 3 will define the functional roles of skeletal regulatory genes in the development of the ossified skeleton using loss- of-function and gain-of-function experiments in three-spine stickleback. Stickleback, a model species related to our Antarctic fish, has a completely sequenced genome, and is amenable to gene knockdown and transgenesis in the laboratory. Significance. These experiments will reveal the identities and functions of genes whose activities have changed, under the force of natural selection, to reduce skeletal ossification in Antarctic fish. Because the reduction of bone mineralization over evolutionary time mimics human bone loss diseases over developmental time, these studies have the potential to identify new genes, and provide new insights into mechanisms for osteopenia, osteoporosis, and other bone wasting disorders that can be exploited to develop novel therapies for human disease. Project Narrative The proposed experiments will reveal the identities and functions of genes whose activities have changed, under the force of natural selection, leading to loss of bone mineral density in certain lineages of Antarctic fish. Because the reduction of bone mineralization over evolutionary time in Antarctic fish mimics the reduction of bone density in humans as they age over developmental time, the proposed studies have the potential to identify new genes, and provide new insights into mechanisms for low bone mineral density, osteoporosis, and other bone wasting disorders that can be exploited to develop novel therapies for human disease.